Project Summary Atopic dermatitis (AD) is a chronic pruritic inflammatory skin disease with an increasing prevalence. Although the etiology of AD is not completely understood, numerous studies suggest that impaired skin barrier function and immune dysregulation underlie the disease. We have developed novel allergen-inducible and spontaneous mouse models that mimic human AD: AD-like skin lesions induced by epicutaneous treatment with allergen require mast cells and the receptor for thymic stromal lymphopoietin (TSLP). We found that phospholipase C (PLC)-3-deficient mice spontaneously develop AD-like skin lesions with increased expression of TSLP and periostin in a mast cell-dependent manner; allergen-induced dermatitis was more severe in PLC-3-/- than in wild-type mice. Gene expression profiles in lesional skin exhibited high similarity between allergen-induced dermatitis, spontaneous dermatitis in PLC-3-/- mice, and human AD. We recently reported that PLC-3 inhibits the proliferation of hematopoietic cells, including mast cells, through formation of the multi-molecular SPS complex containing SHP-1 (protein phosphatase), PLC-3, and Stat5 (transcription factor); PLC-3 interacts with SHP-1 and Stat5, augments the dephosphorylating activity of SHP-1 toward Stat5, and inhibits Stat5 activity. Stat5 and Jak2 (a Stat5-activating kinase) are essential for the development and proliferation of mast cells. Our preliminary data indicated the involvement of mast-cell Stat5 and SHP-1 in AD pathogenesis, as revealed by reduced or enhanced severity of allergen-induced dermatitis in mice with mast cell-specific deletion of Stat5 or SHP-1, respectively, as well as strong efficacy of a Jak-Stat5 inhibitor to prevent allergen-induced dermatitis. Moreover, human AD skin had increased numbers of mast cells with high activation levels of STAT5 and the genes encoding the SPS components were linked to the risk of human AD and/or its severity. These and other preliminary data have led to the following hypotheses: (1) The Stat5- regulatory pathway in mast cells plays a critical pathogenic role in AD. (2) SPS components regulate the activity of the cellular elements of the dermatitis-amplifying/perpetuating vicious cycle consisting of Th2 cytokines (mast cells), periostin (fibroblasts), and TSLP (keratinocytes). (3) The SPS components regulate not only mouse, but also human mast cell biology. Therefore, (Aim 1) using conditional knockout mice of SPS component genes as well as pharmacological approaches, we will further evaluate the role of the mast cell Stat5-regulatory pathway in development and persistence of dermatitis in PLC-3-/- mice. (Aim 2) Using in vitro cultures and in vivo models, we will study how SPS components regulate Th2 cytokine production from mast cells, periostin production from fibroblasts, and TSLP production from keratinocytes. (Aim 3) Finally, we will study the role of SPS components in the growth, survival, and FcRI (high-affinity IgE receptor)-mediated activation of human mast cells and compare mast cells between human AD and healthy individuals. Overall, this project will likely establish a novel paradigm of AD pathogenesis and provide a novel therapeutic strategy.